Although the Warburg effect has long been known as the hallmark characteristic of cancer cells, its exploitation for cancer therapy has never been successful because of failure in identifying an appropriate target protein. Recent studies suggested PFKFB3, which causes a massive increase in F-2,6-P2, the most potent activator of mammalian glycolysis, as the most causative protein of the Warburg effect. We hypothesized that a drug-like PFKFB3 inhibitor will prevent onset of the cancer-specific glycolysis by preventing the F-2,6-P2 surge and, eventually, induce apoptic death of cancer cells. To test this hypothesis, we propose to develop a drug-like PFKFB3 inhibitor. To obtain the molecular basis of the target, which is necessary for design of the desired inhibitor, we determined the crystal structure of human PFKFB3 and suggested a molecular model of its catalytic mechanism. We also performed an initial drug discovery study and obtained several promising PFKFB3 inhibitor molecules. Taking advantage of the progress we have made, we now initiate a full-scale study to develop a drug-like PFKFB3 inhibitor and test if it can cause inhibition of cancer glycolysis and, ultimately, apoptic death of cancer cells. To obtain the molecular templates for PFKFB3 inhibitors, virtual screening and subsequent functional screening of database compounds will be carried out. Hit compounds will be selected on the bases of binding affinity, selectivity, amenability to chemical modification, and structural novelty. The selected hits will be optimized to be drug-like via chemical modification. During this optimization step, new molecules will be synthesized from the hit templates and tested regarding their potency, cell- permeability, stability, and target-selectivity on cultured cancer cell lines. The compounds selected from this test would be considered as the drug-like PFKFB3 inhibitors. Finally, the biochemical and pharmacological properties of these final lead molecules will be investigated to test our hypothesis. This project is aimed to develop a novel drug-like compound with potential of anti-cancer drug. This compound will be designed to inhibit cancer glycolysis, which has never successfully exploited for cancer therapy. Thus, successful completion of this project will have an immediate impact on development of new class cancer drug and new strategies in cancer therapy. PUBLIC HEALTH RELEVANCE: This project is aimed to develop a novel drug-like compound with potential of anti-cancer drug. This compound will be designed to inhibit cancer glycolysis, which has never successfully exploited for cancer therapy. Thus, successful completion of this project will have an immediate impact on development of new class cancer drug and new strategies in cancer therapy.